TET2 is one of the most commonly mutated genes in adult myeloid malignancies and in normal individuals over 70 years of age. Tet2-loss leads to increased expansion of hematopoietic stem/progenitor cells (HSPCs) and subsequent development of myeloid malignancies in mice. Thus, TET2 mutations constitute a suitable target for intervention at the early stages of HSPC clonal expansion. To exploit TET2 mutations therapeutically, a better understanding of the mechanisms by which Tet2-loss leads to premalignant HSPC dysregulation and myeloid malignancy development is essential. As a member of the methylcytosine dioxygenase family, TET2 converts 5mC to 5hmC, which can be relatively stable or be further oxidized to 5fC and 5caC. We have reported the requirement of catalytic activity of TET2 in its tumor-suppressive functions in HSPCs. Recently, we identified a novel mechanism of hematological malignancy via the increased mutational burden at genomic sites where TET2 binds and, counterintuitively, with gained 5hmC upon TET2-loss. In addition, we discovered a novel role of TET2 in RNA hydroxymethylation (hm5C) and target transcripts destabilization via its partner and RNA-binding protein (RBP) PSPC1 in mouse embryonic stem cells. Importantly, we found that PSPC1 and its heterodimer partner NONO are both abundantly expressed in HSPCs. Our studies raise two critical questions on TET2 biology in normal and malignant hematopoiesis: 1) How does gained 5hmC contribute to the pathogenesis of myeloid malignancy? 2) Is catalytic activity of TET2 relevant and functional at the RNA hm5C level in HSPCs? We hypothesize that TET2 may exert enzymatic functions through preferential 5hmC- to-5fC/5caC oxidation leading to DNA demethylation and transcriptional activation of tumor suppressor genes and through RNA hm5C modification and consequent destabilization of oncogenic RNAs. We will study how dysregulation of DNA-5hmC and RNA-hm5C mediated epigenetic control upon TET2 mutations can lead to malignant hematopoiesis by three specific aims. Aim 1) Define preferential TET2-mediated 5hmC-5fC/5caC oxidation for DNA demethylation in HSPCs and its dysregulation for the pathogenesis of myeloid malignancy using our newly created 5hmC stalling and catalytically inactive knock-in mutant Tet2 mouse models. Aim 2) Determine the functional significance of the TET2-RUNX1 partnership for TET2 genomic localization and target gene expression in HSPCs. RUNX1, a TET2 binding partner, is reported to be enriched in regions of increased 5hmC, but not in hypo-5hmC regions in TET2 mutant AML. We will study RUNX1 functions in regulating TET2 genomic occupancy in HSPCs and examine the potential role of RUNX1 in imparting unique TET2 functions in preferential 5hmC-to-5fC/5caC oxidation during normal and malignant hematopoiesis. Aim 3) Investigate RBP- mediated TET2 recruitment for RNA hm5C modification and destabilization of oncogenic RNAs in HSPCs and its dysregulation in hematological malignancy. We will also investigate how mutation of TET2 may impair its PSPC1 association in HSPCs leading to malignant hematopoiesis.